MicroRNA-132 enhances transition from inflammation to proliferation during wound healing
Dongqing Li, Aoxue Wang, Xi Liu, Florian Meisgen, Jacob Grünler, Ileana R. Botusan, Sampath Narayanan, Erdem Erikci, Xi Li, Lennart Blomqvist, Lei Du, Andor Pivarcsi, Enikö Sonkoly, Kamal Chowdhury, Sergiu-Bogdan Catrina, Mona Ståhle, Ning Xu Landén
Dongqing Li, Aoxue Wang, Xi Liu, Florian Meisgen, Jacob Grünler, Ileana R. Botusan, Sampath Narayanan, Erdem Erikci, Xi Li, Lennart Blomqvist, Lei Du, Andor Pivarcsi, Enikö Sonkoly, Kamal Chowdhury, Sergiu-Bogdan Catrina, Mona Ståhle, Ning Xu Landén
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Research Article Inflammation

MicroRNA-132 enhances transition from inflammation to proliferation during wound healing

Abstract

Wound healing is a complex process that is characterized by an initial inflammatory phase followed by a proliferative phase. This transition is a critical regulatory point; however, the factors that mediate this process are not fully understood. Here, we evaluated microRNAs (miRs) in skin wound healing and characterized the dynamic change of the miRNome in human skin wounds. miR-132 was highly upregulated during the inflammatory phase of wound repair, predominantly expressed in epidermal keratinocytes, and peaked in the subsequent proliferative phase. TGF-β1 and TGF-β2 induced miR-132 expression in keratinocytes, and transcriptome analysis of these cells revealed that miR-132 regulates a large number of immune response– and cell cycle–related genes. In keratinocytes, miR-132 decreased the production of chemokines and the capability to attract leukocytes by suppressing the NF-κB pathway. Conversely, miR-132 increased activity of the STAT3 and ERK pathways, thereby promoting keratinocyte growth. Silencing of the miR-132 target heparin-binding EGF-like growth factor (HB-EGF) phenocopied miR-132 overexpression in keratinocytes. Using mouse and human ex vivo wound models, we found that miR-132 blockade delayed healing, which was accompanied by severe inflammation and deficient keratinocyte proliferation. Together, our results indicate that miR-132 is a critical regulator of skin wound healing that facilitates the transition from the inflammatory to the proliferative phase.

Authors

Dongqing Li, Aoxue Wang, Xi Liu, Florian Meisgen, Jacob Grünler, Ileana R. Botusan, Sampath Narayanan, Erdem Erikci, Xi Li, Lennart Blomqvist, Lei Du, Andor Pivarcsi, Enikö Sonkoly, Kamal Chowdhury, Sergiu-Bogdan Catrina, Mona Ståhle, Ning Xu Landén

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Figure 4

miR-132 regulates keratinocyte chemokine production by suppressing NF-κB signaling.

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miR-132 regulates keratinocyte chemokine production by suppressing NF-κB...
(A) qRT-PCR analysis of chemokine expression in keratinocytes transfected with pre-miR-132 or pre-miR-Ctrl for 48 hours and then treated with TNF-α for 24 hours (n = 3). (B) HUVECs were incubated with conditioned medium from keratinocytes treated as above, and expression of endothelial cell activation markers was analyzed by qRT-PCR (n = 3). (C) Human neutrophil chemotaxis toward the conditioned medium from keratinocytes treated as above. Flow cytometric plots show forward scatter/side scatter (FSC/SSC) of the migrated cells. (D) Genes in microarray data were ranked by fold change (pre-miR-132/pre-miR-Ctrl). GSEA evaluated enrichment within the profile data for the reported target genes of the NF-κB pathway. Vertical bars along the x axis denote the positions of the NF-κB target genes within the ranked list. NES, normalized enrichment score. (E) Luciferase activity was measured in keratinocytes transfected with NF-κB reporter and pre-miR-132 or pre-miR-Ctrl for 24 hours and treated with TNF-α for 5 hours (n = 3). Keratinocytes transfected with pre-miR-132 or pre-miR-Ctrl for 48 hours were treated with TNF-α for 5 to 80 minutes. Phosphorylated and total IκB (F) and phosphorylated and total p65 (G) were detected in total protein lysates; p65 was detected in the nuclear lysates by Western blotting; and PARP was detected on the same blots as the loading control (H). (I) Immunostaining of p65 in keratinocytes transfected with pre-miR-132 or pre-miR-Ctrl for 48 hours then treated with TNF-α for 20 minutes. Cells were counterstained using DAPI. *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001 by Student’s t test.